The individual reactions of the lipid-linked saccharide pathway are being investigated in order to understand the assembly of the oligosaccharide portion of membrane glycoproteins. Since these reactions are all mediated by membrane bound glycosyl transferases, the specific reactions can only be studied in solubilized systems. Using Triton X-100, we have solubilized a G1cNAc transferase which adds the second G1cNAc to form N,N'-diacetylchitobiosyl-pyrophoshoryl-dolichol and a mannosyl transferase that adds the first mannose to form mannosyl-G1cNAc-G1cNAc-pyrophosphoryl-dolichol. These enzymes are currently being purified and characterized. In the partially purified state, the enzymes are activated by an unidentified factor which elutes from columns in a different fraction than the enzyme. Attempts to identify this activator are in progress. Soybean cells in culture secrete a glycoprotein into the medium which can be labeled with radioactive mannose. The incorporation of radioactive mannose into this protein is inhibited by the addition of tunicamycin to these cells indicating that the carbohydrate is of the glycosylamine linkage. However, incorporation of leucine is not blocked by this antibiotic. Studies are in progress to isolate and characterize the protein.